Novel aminoglycoside antibiotics, process for producing the same, and pharmaceutical use thereof

ABSTRACT

The present invention relates to novel aminoglycoside antibiotics, a process for producing the same, and pharmaceutical use thereof. More specifically, the present invention relates to compounds represented by formula (I), a process for producing the same, and use of the same as antimicrobial agents. 
     
       
         
         
             
             
         
       
     
     wherein R represents amino or hydroxyl.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based upon and claims the benefit of priority fromthe prior Japanese Patent Application No. 310618/2007, filed on Nov. 30,2007, the entire contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates to novel aminoglycoside antibiotics, aprocess for producing the same, and pharmaceutical use thereof.

BACKGROUND ART

Aminoglycoside antibiotics are a generic term for glycoside antibioticscontaining amino sugar or aminocyclitol and exclude a group ofantibiotics such as macrolides, nucleosides, and anthracyclines. Up tonow, a number of aminoglycoside antibiotics have been discovered fromculture of actinomyces or bacteria. Among them, streptomycin, neomycin,kanamycin, gentamicin, ribostamycin, tobramycin and the like have beenused as useful chemotherapeutic agents. On the other hand, thewidespread use of these aminoglycoside antibiotics in clinical practicehas led to a problem of the appearance of bacteria resistant toaminoglycoside antibiotics.

Kanamycins (kanamycin A, kanamycin B, and kanamycin C) areaminoglycoside antibiotics produced by Streptomyces kanamyceticus.Kanamycins have a wide spectrum of antimicrobial activity. Since,however, many infecting bacteria become rapidly resistant to kenamycins,in recent years, the clinical adaptation of kanamycins is limited todiseases, mainly tuberculosis.

In kanamycins, kanamycin derivatives such as dibekacins, amicacins, andarbekacins effective also against resistant bacteria have been developedbased on studies on a resistant mechanism. However, bacteria resistantto these agents are appearing. Under such circumstances, the developmentof novel aminoglycoside antibiotics that are effective against resistantbacteria and can reduce nephrotoxicity that is a problem common toaminoglycoside antibiotics has been expected.

Regarding aminoglycoside antibiotics comprising 2-deoxystreptamine asone constituent sugar, studies have been made on the production of novelaminoglycoside antibiotics by acquiring a mutant strain which producesan aminoglycoside antibiotic 2-deoxystreptamine-dependently, adding a2-deoxystreptamine analog to the mutant strain, and cultivating themixture. Also in kanamycins, there is a report that antibioticsdifferent from kanamycins are produced by acquiring a mutant strainhaving a phenotype of 2-deoxystreptamine-dependent kanamycin production,adding 2-epistreptamine to the mutant strain, and cultivating themixture (U.S. Pat. No. 3,669,838). Further, there is a report that4-O-(α-D-glucopyranosyl)6-O-(3-amino-3-deoxy-α-D-glucopyranosyl)1-N-methyl-2-deoxystreptamineor4-O-(α-D-glucopyranosyl)6-O-(3-amino-3-deoxy-α-D-glucopyranosyl)2-epi-streptamineis produced by adding 1-N-methyl-deoxystreptamine ormyo-inosadiamine-1,3(2-epistreptamine) to a mutant strain having aphenotype of 2-deoxystreptamine-dependent kanamycin production andcultivating the mixture (Kojima, M. and Satoh, A., “Journal ofAntibiotics”, (Japan), 1973, Vol. 26, p. 784-786). Furthermore, there isa report that4-O-(6-amino-6-deoxy-α-D-glucopyranosyl)6-O-(3-amino-3-deoxy-α-D-glucopyranosyl)streptamine(LL-BM27α) and4-O-(6-amino-6-deoxy-α-D-glucopyranosyl)6-O-(α-D-glucopyranosyl)streptamine(LL-BM2713) are produced by adding streptamine to a mutant strain havinga phenotype of 2-deoxystreptamine-dependent kanamycin production andcultivating the mixture (Borders, D. B. et al., “Journal ofAntibiotics”, (Japan), 1982, Vol. 35, p. 1107-1110). Here LL-BM27α issynonymous with 2-hydroxykanamycin A.

The amounts of aminoglycoside antibiotics produced by the addition ofthe substances and cultivation of the mixture are so small that theindustrial applicability of the aminoglycoside antibiotics is low.Accordingly, it can be said that novel aminoglycoside antibiotics whichare clinically useful and have potent antimicrobial activity are stilldemanded.

SUMMARY OF THE INVENTION

The present inventors have now found that novel aminoglycosideantibiotics having potent antimicrobial activity can be produced bycultivating a kanamycin producing strain derived from the genusStreptomycin together with a specific 2-deoxystreptamine analog. Thepresent invention has been made based on such finding.

Accordingly, an object of the present invention is to provide novelaminoglycoside antibiotic agents possessing potent antimicrobialactivity and a process for producing the same.

According to the present invention, there are provided aminoglycosideantibiotics that are compounds represented by formula (I) or theirpharmacologically acceptable salts or their solvates.

wherein R represents amino or hydroxyl.

According to another aspect of the present invention, there is provideda process for producing compounds represented by formula (I), theprocess comprising cultivating a kanamycin producing strain of the genusStreptomyces in a medium which comprises streptamine and/or myo-inositolto produce the compound.

The compounds according to the present invention have potentantimicrobial activity against bacteria causative of various infectiousdiseases and can be advantageously utilized in the treatment ofinfectious diseases. Further, the production process according to thepresent invention can simply and stably supply the above compounds.

DETAILED DESCRIPTION OF THE INVENTION Deposit

The strain S. Kanamyceticus-DOS according to the present invention hasbeen deposited with International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology (address:Tsukuba Central 6 Tsukuba-shi, Higashi 1-1-1, Ibaraki, 305-8566 Japan)(original deposited date: Nov. 1, 2007) under accession number FERMBP-11052.

DEFINITION

The term “2-hydroxykanamycin A” as used herein refers to a compoundhaving hydroxyl introduced into the 2-position of kanamycin A. The term“2-hydroxykanamycin” refers to a group of compounds having hydroxylintroduced into the 2-position of kanamycins (kanamycin A, kanamycin B,and kanamycin C). Specifically, the group of compounds includes2-hydroxykanamycin A, 2-hydroxykanamycin B, and 2-hydroxykanamycin C.

Further, the kanamycin producing strain, as used herein, which is“deoxystreptamine dependent” refers to a mutant strain, among kanamycinproducing bacteria, that can restore the capability to producekanamycins by adding deoxystreptamine.

The polypeptide “having activity functionally equivalent to” or “havingfunctionally equivalent activity” as used herein refers to the followingpolypeptide.

In the polypeptide, in addition to polymorphisms or mutants of genescoding for the polypeptide, structural mutants may occur in an aminoacid sequence, for example, by a modification reaction. However, it isknown that some polypeptides, despite the presence of these mutants,have substantially the same physiological and biological activity aspolypeptides not having a mutant. The polypeptide which, despite thestructural difference, does not have a large difference in functionrefers to a polypeptide “having functionally equivalent activity.”

Compounds Represented by Formula (I) or their PharmacologicallyAcceptable Salts or their Solvates

One characteristic feature of the compounds represented by formula (I)according to the present invention is that hydroxyl has been introducedinto the 2-position of kanamycin B or C. The compounds having thestructure have a broad antimicrobial spectrum ranging from gram-positivebacteria to gram-negative bacteria including Pseudomonas aeruginosa andhave potent antimicrobial activity.

According to one embodiment of the present invention, in the compoundsrepresented by formula (I), R represents amino. This compound(hereinafter referred to as “hydroxykanamycin B”) has a structurerepresented by formula (1).

According to another embodiment of the present invention, in thecompounds represented by formula (I), R represents hydroxyl. Thiscompound (hereinafter referred to as “2-hydroxykanamycin C”) has astructure represented by formula (2).

The compounds represented by formula (I) may be present as salts. Suchsalts include, for example, pharmaceutically acceptable salts. Specificexamples thereof include hydrohalides such as hydrofluorides,hydrochlorides, hydrobromides, or hydroiodides, inorganic acid saltssuch as sulfates, phosphates, perchlorates, or carbonates, salts ofcarboxylic acids such as acetic acid, trichloroacetic acid,trifluoroacetic acid, hydroxyacetic acid, lactic acid, citric acid,tartaric acid, oxalic acid, benzoic acid, mandelic acid, butyric acid,maleic acid, propionic acid, formic acid, or malic acid, salts of aminoacids such as alginic acid, aspartic acid, or glutamic acid, or salts ofsulfonic acid such as methanesulfonic acid or p-toluenesulfonic acid.Preferred are hydrohalides such as hydrochlorides and inorganic acidsalts such as sulfates.

The compounds represented by formula (I) or its pharmaceuticallyacceptable salts may exist as their solvates. Preferred solvates includehydrates, methanolates, or ethanolates.

Producing Strains

The compounds represented by formula (I) can be produced by variousmethods. For example, as described above, the compounds represented byformula (I) can be produced by cultivating a kanamycin producing strainof the genus Streptomyces in a medium comprising streptamine and/ormyo-inositol.

Examples of such suitable producing strains includedeoxystreptamine-dependent kanamycin producing strains. More preferredare strains of the genus Streptomyces wherein 2-deoxy-scyllo-inososesynthase catalyzing a first reaction in 2-deoxystreptamine biosynthesisfrom glucose-6-phosphoric acid has been inactivated. It is surprisingthat, when such strains are cultivated together with a deoxystreptamineanalog, the compounds according to the present invention can beselectively produced without producing kanamycins.

Accordingly, according to still another aspect of the present invention,there is provided a kanamycin producing strain of the genus Streptomycescapable of producing compounds represented by formula (I) wherein2-deoxy-scyllo-inosose synthase has been inactivated.

The mutant producing strain can be acquired, for example, by treatingkanamycin producing bacteria which are derived from the genusStreptomyces including Streptomyces kanamyceticus, for example, byartificial mutation methods including ultraviolet (UV) irradiation ornitrosoguanidine (NTG). The acquisition of the desired mutant strain canbe confirmed, for example, by acquiring a deoxystreptamine-dependentkanamycin producing strain, then measuring the activity of intracellular2-deoxy-scyllo-inosose synthase in each mutant strain by a conventionalmethod (Kudo, F. et al., “Journal of Antibiotics”, (Japan), 1999, Vol.52, p. 81-88), and selecting a mutant strain that does not have enzymeactivity.

Regarding the kanamycin producing strain in which 2-deoxy-scyllo-inososesynthase has been inactivated, genes coding for 2-deoxy-scyllo-inososesynthase have already been clarified (Japanese Patent ApplicationLaid-Open No. 173537/2004). Accordingly, a desired mutant strain canalso be acquired by gene recombination technology. For example, adesired mutant strain may be acquired by destroying a gene coding for2-deoxy-scyllo-inosose synthase. Further, the mutant strain can also beacquired by preparing a mutant gene having an amino acid substitutionthat provides inert 2-deoxy-scyllo-inosose synthase, and subjecting themutant gene to gene substitution with a wild-type gene on a chromosome.The gene can be destroyed or substituted by methods commonly used inActinomyces (“Practical Streptomyces Genetics”, “The John InnesFoundation”, (England), Norwick, 2000, p. 311-338). The disclosures ofthe document are incorporated herein by reference.

For example, a mutation in which aspartic acid at position 136 of theamino acid sequence of the synthase represented by SEQ ID No. 1 issubstituted by asparagines may be mentioned as one example of mutationby which 2-deoxy-scyllo-inosose synthase is inactivated. Accordingly,the strain that can produce the compound represented by formula (I) canbe acquired by integrating a gene coding a polypeptide having themutation or a gene coding for an analog functionally equivalent to thepolypeptide into a strain.

According to a preferred embodiment of the present invention, thekanamycin producing strain capable of producing the compound representedby formula (I) is the strain into which a gene coding for a polypeptideselected from the following polypeptides (a) to (c) has been integrated:

(a) a polypeptide consisting of an amino acid sequence represented bySEQ ID No. 1 having a mutation in which aspartic acid at position 136has been changed to asparagine,

(b) a polypeptide consisting of the amino acid sequence defined in (a)wherein one or more amino acids have been substituted, deleted, added,or inserted, the polypeptide having an activity functionally equivalentto the polypeptide defined in (a), and

(c) a polypeptide consisting of an amino acid sequence having 80% ormore homology with the amino acid sequence defined in (a), thepolypeptide having an activity functionally equivalent to thepolypeptide defined in (a)

S. Kanamyceticus-DOS may be mentioned as an example of suitablekanamycin producing strains corresponding to (a).

In (b), the “one or more amino acids” is preferably 1 to 40 amino acids,more preferably 1 to 8 amino acids, still more preferably 1 to 4 aminoacids.

In (d), the homology is preferably not less than 90%, more preferablynot less than 95%.

Further, when stable production of the compound represented by formula(I) is taken into consideration, preferably, the polypeptide describedin (b) or (c) holds a mutation in which aspartic acid at position 136 inthe amino acid sequence described in (a) or at a position correspondingto the position 136 has been changed to asparagine. The presence orabsence of the mutation in the amino acid sequence described in (a) to(c) and the determination of sequence homology can be properlydetermined by a person having ordinary skill in the art by comparing theamino acid sequence represented by SEQ ID No. 1 with the amino acidsequence in (a) to (c) by a conventional method.

The functional equivalency between the polypeptide described in (b) or(c) and the polypeptide described in (a) can be confirmed by measuring2-deoxy-scyllo-inosose synthase activity for both the polypeptidesaccording to the above-described method described in Kudo, F. et al.,“Journal of Antibiotics”, (Japan), 1999, Vol. 52, p. 81-88 and comparingthe measured results. Further, the functional equivalency can beindirectly confirmed by measuring the deoxystreptamine dependency orantimicrobial activity of kanamycin producing strain with genes codingfor the polypeptides integrated thereinto by a method described inExample 2 or Test Example 1 and statistically comparing the measuredresults.

Production Process

As described above, the compound represented by formula (I) according tothe present invention can be produced by cultivating a kanamycinproducing strain derived from the genus Streptomycin in a medium whichcomprises a 2-deoxystreptamine analog selected from streptamine andmyo-inositol.

When selective production of the compound represented by formula (I) istaken into consideration, as described above, the kanamycin producingstrain derived from the genus Streptomycin is preferably a kanamycinproducing strain in which 2-deoxy-scyllo-inosose synthase has beeninactivated. However, a kanamycin producing strain in which2-deoxy-scyllo-inosose synthase has not been inactivated may also beused. Examples of suitable strains in which 2-deoxy-scyllo-inososesynthase has not been inactived include Streptomyces kanamyceticus.

Further, in the production process according to the present invention, acombination of the kanamycin producing strain with the2-deoxystreptamine analog added to the medium may be properly determinedby taking the type of the desired contemplated compound intoconsideration.

According to one embodiment of the present invention, the kanamycinproducing strain is one in which 2-deoxy-scyllo-inosose synthase hasbeen inactivated, and the medium comprises streptamine. According tothis embodiment, the compound represented by formula (I) can be producedtogether with 2-hydroxykanamycin A. This is advantageous in that2-hydroxykanamycins (2-hydroxykanamycins A to C) can be simultaneouslyproduced. Further, according to another embodiment of the presentinvention, the kanamycin producing strain is one in which2-deoxy-scyllo-inosose synthase has been inactivated, and the mediumcomprises myo-inositol. According to this embodiment, 2-hydroxykanamycinC, which is a compound represented by formula (I) wherein R representshydroxyl, can be selectively produced.

The addition amount of streptamine or myo-inositol may be properlyvaried depending upon cultivation conditions, but is preferably 100 to50,000 μg/ml, more preferably 1,000 to 10,000 μg/ml.

Conventional substances may be properly added as ingredients of themedium in the production process according to the present invention.

Medium ingredients other than streptamine and myo-inositol usable hereininclude, for example, carbon sources such as glucose, sucrose, starchsyrups, dextrins, starches, glycerol, syrups, animal and vegetable oils;and nitrogen sources such as soybean meals, wheat germ oils, corn steepliquors, cottonseed cakes, meat extracts, polypeptone, malt extracts,yeast extracts, ammonium sulfate, sodium nitrate, and urea. Further, ifnecessary, the addition of sodium, potassium, calcium, magnesium,cobalt, chlorine, phosphoric acid (for example, dipotassiumhydrogenphosphate), sulfuric acid (for example, magnesium sulfate) andinorganic salts which can produce other ions is also effective.Furthermore, if necessary, various vitamins such as thiamines (forexample, thiamine hydrochloride), glutamic acid (for example, sodiumglutamate), amino acids such as asparagine (for example, DL-asparagine),micronutrients such as nucleotides, and selected drugs such asantibiotics may also be added. Furthermore, organic and inorganicsubstances that aid the growth of bacteria and promote the production of2-hydroxykanamycin B and 2-hydroxykanamycin C can be properly added.

The medium is preferably approximately pH 5.5 to pH 9.

The cultivation may be carried out by solid cultivation, shakingcultivation, aeration agitation cultivation, or deep aerobic cultivationunder aerobic conditions. Among them, the deep aerobic cultivation ispreferred.

For example, 15° C. to 40° C. is suitable as the cultivationtemperature. In many cases, however, bacteria are grown at a temperaturearound 25° C. to 35° C.

The amount of the compound represented by formula (I) produced variesdepending upon the medium, cultivation conditions, and cultivationmethod used, but reaches the largest, for example, 2 days to 15 days.

Preferably, when the amount of the compound represented by formula (I)in the medium has reached the largest, the cultivation is stopped. Thecompound is then harvested from the culture and is purified.

In order to harvest the compound of the present invention from theculture, a conventional separation means utilizing the properties of thecompound may be used. The separation means may be, for example, solventextraction, ion exchange resin methods, adsorption or partition columnchromatography, gel filtration, dialysis, precipitation, andcrystallization, either alone or in an appropriate combination. Forexample, the culture is filtered to give a filtrate. The filtrate isthen adsorbed on a cation exchange resin such as Amberlite IRC-50 orAmberlite FPC3500, and elution is carried out with aqueous ammonia. Theeluate is further purified with a cation exchange resin such as Dowex50W or Amberlite CG-50 and is if necessary purified by ion-exclusionchromatography with Dowex 1 or by adsorption chromatography with HP20ss,whereby each 2-hydroxykanamycin represented by formula (I) can beisolated.

Use

The compounds according to the present invention have potentantimicrobial activity and are useful for administration as a medicinesto animals including human. Thus, according to a further aspect of thepresent invention, there is provided a pharmaceutical compositioncomprising a compound represented by formula (I) or itspharmacologically acceptable salt or their solvates. The composition ispreferably used as an antimicrobial agent.

According to another aspect of the present invention, there is provideduse of a compound represented by formula (I) or its pharmacologicallyacceptable salt or their solvates, in the manufacture of apharmaceutical composition. According to still another aspect of thepresent invention, there is provided use of a compound represented byformula (I) or its pharmacologically acceptable salt or their solvates,in the manufacture of an antimicrobial agent.

When the compound according to the present invention is used as apharmaceutical composition, the pharmaceutical composition may beformulated according to various dosage forms or usage forms byconventional methods. Pharmaceutical preparations for oraladministration include tablets, pills, granules, capsules, powders,liquid formulations, suspensions, syrups, and sublingual agents.Pharmaceutical preparations for parenteral administration includeinjections, transdermal agents, inhalants, and suppositories.Pharmaceutical additives such as surfactants, excipients, stabilizers,wetting agents, disintegrants, dissolution aids, tonicity adjustingagents, buffers, colorants, and flavoring agents are properly used inthe formulation.

Pharmaceutically acceptable carriers may be used as the carrier for thepharmaceutical composition. The type and composition of the carrier maybe properly determined according to administration routes andadministration methods. For example, liquid carriers usable hereininclude water, alcohols, soybean oils, and sesame oils. Example of solidcarriers include sugars such as maltose and sucrose, amino acid saltssuch as lysine, polysaccharides such as cyclodextrin, organic acid saltssuch as magnesium stearate, and cellulose derivates such ashydroxylpropyl cellulose.

The compounds according to the present invention having antimicrobialactivity are preferably used in the treatment or prevention ofinfectious diseases. Accordingly, according to a further aspect of thepresent invention, there is provided a method for treating or preventingan infectious disease, comprising administering an effective amount of acompound represented by formula (I) or its pharmacologically acceptablesalt or their solvates to an animal including human. The term“treatment” as used herein means ameliorating an established diseasestate, and the term “prevention” as used herein means preventing theestablishment of a disease state in the future.

The compounds according to the present invention can be applied tobacteria causative of various infectious diseases. Bacteria causative ofinfectious diseases include, for example, Staphylococcus aureus,Staphylococcus epidermidis, Entercoccus, Escherichia coli, Pseudomonasaeruginosa, Bacillus subtilis, Salmonella, or Acinetobacter. Preferredis Staphylococcus aureus, Escherichia coli, or Pseudomonas aeruginosa.

When the compound represented by formula (I) is 2-hydroxykanamycin B,the bacteria causative of infectious diseases are preferablyStaphylococcus aureus, Staphylococcus epidermidis, Entercoccus,Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Salmonella,or Acinetobacter, more preferably Staphylococcus aureus, Staphylococcusepidermidis, Entercoccus, Escherichia coli, Bacillus subtilis,Salmonella, or Acinetobacter, still more preferably Staphylococcusaureus, Staphylococcus epidermidis, Escherichia coli, Bacillus subtilis,Salmonella, or Acinetobacter.

When the compound represented by formula (I) is 2-hydroxykanamycin C,the bacteria causative of infectious diseases are preferablyStaphylococcus aureus, Staphylococcus epidermidis, Escherichia coli,Bacillus subtilis, Salmonella, or Acinetobacter, more preferablyStaphylococcus epidermidis, Escherichia coli, Bacillus subtilis,Salmonella, or Acinetobacter, still more preferably Staphylococcusepidermidis, Bacillus subtilis, Salmonella, or Acinetobacter.

The effective dose of the compound according to the present inventionmay be properly determined by physicians in consideration of particularconditions, for example, the age, weight, type and severity of patients,and administration route. When the compound is orally administered tohuman, for example, the compound can be administered, for example, at adose of 0.01 to 1000 mg/kg per adult per day. On the other hand, whenthe compound is intravenously administered, the compound can beadministered at a dose of 0.001 to 100 mg/kg per adult per day.

EXAMPLES

The present invention is further illustrated by the following Examplesthat are not intended as a limitation of the invention.

In the following Examples, LC/MS analyses were carried out under thefollowing conditions.

Conditions for LC/MS Analyses

(HPLC part: Waters 2690)

Column: Capcell Pak C18 MG, 4.6×150 mm, 5 μm (manufactured by ShiseidoCompany, Limited)

Mobile phase: A: 0.2% Aqueous pentafluoropropionic acid solution

B: Acetonitrile

C: H₂O

Liner gradient: 0 min (A/B/C=10/10/80)→15 min (A/B/C=10/30/60)

Flow rate: 0.4 ml/min, temp.: 30° C.

(MS part: Waters ZQ) ESI method

Ion source temp.: 100° C.

Desolvation temp.: 380° C.

Desolvation gas flow: 350 L/hr

Cone gas flow: 50 L/hr

Capillary voltage: 3.5 kV

Cone voltage: positive: 30 V

Example 1 Construction of Plasmid pDDOI for Introduction of2-Deoxy-Scyllo-Inosose Synthase Gene (Orf11) Mutation of Streptomyceskanamyceticus

Regarding 2-deoxy-scyllo-inosose synthase (SEQ ID No. 1), aspartic acidat position 136 conserved as an amino acid residue important to bindingto a substrate, glucose-6-phosphoric acid, was changed to asparagine toinactivate enzyme protein. Preparation was carried out by a PCR reactionusing pKM9 (see Japanese Patent Application Laid-Open No. 173537/2004,Example 2, FERM P-19117) as a template.

A kanamycin biosynthetic cluster region utilized is shown in SEQ ID No.2.

The following primers, i.e., a primer including Hind III or Xba Idigestion site and a primer which had been designed so that asparticacid (GAT) at position 136 from an initiation codon of a DOI synthasegene is mutated to asparagine (AAC), were used.

Km-Mu-Hind III (SEQ ID No. 3) 5′-GGGAAGCTTGACCTTGGAGGTATGTGT-3′ Km-Mu-L(SEQ ID No. 4) 5′-GTTCAGCATGGCCACCACGGTGGT-3′(The underlined part represents mutation introduced part) Km-Mu-R(SEQ ID No. 5) 5′-TCGGTGCTCTCGCTCAAGCAG-3′ Km-Mu-Xba I (SEQ ID No. 6)5′-GGGTCTAGATGCCGTCCTGGTGGTAGT-3′

A PCR reaction was carried out using a primer combination of Km-Mu-HindIII (SEQ ID No. 3) with Km-Mu-L (SEQ ID No. 4) and a primer combinationof Km-Mu-R (SEQ ID No. 5) with Km-Mu-Xba I (SEQ ID No. 6). The reactionwas carried out using about 1 μg of genomic DNA, 0.3 μM of each primer,and KOD plus DNA polymerase (manufactured by TOYOBO CO., LTD.) underconditions of 94° C./2 min (94° C./15 sec, 50° C./30 sec, and 68° C./1.5min)×25 cycles. As a result, about 1.5 kbp DNA fragments werespecifically amplified. The DNA fragments were purified by a QIAquickPCR purification kit (manufactured by QIAGEN K.K.). The blunt end wasphosphorylated (manufactured by NIPPON GENE CO., LTD.), and thephosphorylated DNA fragments were digested with Hind III and Xba I,followed by cloning into Hind III and Xba I sites of pUC119. The basesequence of the cloned DNA fragments were analyzed. As a result, itcould be confirmed that the DNA fragments contained a2-deoxy-scyllo-inosose synthase (orf11) gene (SEQ ID No. 7) withcontemplated substitution by asparagine inserted thereinto.

Plasmid pSET152 (Bierman, M. et al., “Gene”, (Netherlands), 1992, Vol.116, p. 43-49) for conjugation transfer of Actinomyces was digested withSph I and was blunted with T4 DNA polymerase. A Hind III linker(manufactured by TAKARA SHUZO CO., LTD.) was then linked thereto toconstruct pSET153. An about 2.8 kbp Hind III-Xba I fragment derived frompSET153 was linked to an about 3 kbp Hind III-Xba I fragments containingthe gene subjected to amino acid substitution to obtain plasmid pDDOIfor orf11 gene mutation introduction that had a conjugation transferability.

Example 2 Creation of Deoxystreptamine-Non-Producing Strain by PlasmidpDDOI for Orf11 Gene Mutation Introduction

Streptomyces kanamyceticus which is a kanamycin producing bacterium wascoated onto an MS agar medium (2% S soybean meal, 2% mannitol, 2% agar)and was cultivated at 28° C. for 3 days. After the cultivation, hyphaewere scraped with 3 ml of 20% glycerol and were collected to prepare ahypha liquid of a host.

On the other hand, the E. coli (Escherichia coli) ET12567/pUZ8002 straincarrying plasmid pDDOI was inoculated into 100 ml of an LB liquid medium(1% Difco Bacto tryptone, 0.5% Difco yeast extract, 0.5% NaCl, and 0.1%glucose) containing 25 μg/ml of chloramphenicol, 25 μg/ml of kanamycin,and 50 μg/ml of apramycin and was cultivated at 37° C. overnight toprepare a preculture. The culture was inoculated into the same liquidmedium as the precultivation so that the final concentration of thepreculture was 1%, followed by cultivation at 37° C. for about 4 hr. Theculture was washed twice with an LB liquid medium and was finallysuspended in 10 ml of an LB liquid medium to prepare an E. coli liquid.

The hypha liquid of the host (500 μl) prepared above and 500 μl of theE. coli liquid were mixed together for harvesting. The harvestedbacteria were then coated on an MgCl₂-added MS agar medium so that thefinal concentration of the bacteria was 10 mM. After cultivation at 28°C. for 20 hr, 1 ml of sterilized water containing 1 mg of apramycin and1.5 mg of nalidixic acid was overlayered, and the cultivation wascontinued at 28° C. for 5 days to obtain an apramycin-resistant strain.

A genomic DNA was prepared from the apramycin-resistance strain with aMagExtractor genomic DNA extractor (manufactured by TOYOBO CO., LTD.)according to a protocol, and it was confirmed by PCR and an southernblot analysis that pDDOI was inserted into the chromosome by homologousrecombination.

The homologous recombinant was inoculated into a modified YEME medium(50 ml), followed by shake cultivation at 28° C. for 2 days, and 1 ml ofthe culture was further inoculated into a fresh modified YEME medium (50ml) to perform successive cultivation. This procedure was repeated fivetimes. Thereafter, the culture diluted to a suitable vial cell count wascoated onto an MS agar medium, followed by cultivation at 28° C. for 4days. The grown colony was replicated onto an MS agar medium containing20 μg/ml of apramycin and onto an apramycin-free MS agar medium, and 18apramycin-sensitive strains that cannot grow in the apramycin-containingmedium were selected.

Genomic DNAs of the apramycin-sensitive strains were prepared, and a PCRreaction was carried out using a primer combination of Km33(5′-CTTCGTGAATCCCCCTT-3′: SEQ ID No. 8) with Km35(5′-GCCCACCGCCTCGATCA-3′: SEQ ID No. 9) to obtain about 3.5 kbpamplified DNA fragments. The base sequence of these amplified DNAfragments was analyzed. As a result, one strain was a mutant strain inwhich substitution by asparagines as designed was observed, and, for 17strains, the base sequences remained unchanged.

In order to examine the productivity of kanamycin, these strains wereinoculated into 30 ml of a liquid growing medium (Umezawa, H. et al.,“The Journal of Antibiotics”, (Japan), 1977, Vol. 30, p. 181-188)prepared in a 250 ml-volume conical flask, followed by cultivation at28° C. for two days. Thereafter, 1 ml of the culture was inoculated in30 ml of a liquid producing medium (in which the amount of starch wasincreased from 1.2% to 6%), and shake cultivation was carried out at 26°C. for 7 days. In order to analyze the product, the culture was adjustedto pH 2.5 with 50% H₂SO₄, was placed in a 1.5-ml Eppendorf tube, and wascentrifuged under conditions of 17,400×g and 10 min, and the supernatantwas subjected to an LC/MS analysis. As a result, for one orf11 mutationintroduced strain, the production of kanamycins was not observed, and 17strains returned to the same base sequence as the parent strain producedkanamycin A (retention time 8.2 min, m/z 485) and kanamycin B (retentiontime 10.4 min, m/z 484).

Next, the kanamycin non-producing strain was coated onto an agar mediumprepared by adding deoxystreptamine to the liquid medium, diluted to ahalf concentration, so as to give a concentration of 200 μg/ml, followedby cultivation at 28° C. for 7 days. Thereafter, the agar was frozen andthawed to extract the product which was then bioassayed. The assaybacterium was Bacillus subtilis ATCC6633. As a result, the productobtained by deoxystreptamine-free cultivation did not have antimicrobialactivity, whereas, for the product obtained by deoxystreptamine-addedcultivation, an inhibition circle indicating antimicrobial activity wasdetected. Accordingly, the product obtained by deoxystreptamine-addedcultivation was subjected to an LC/MS analysis. As a result, kanamycin Aand kanamycin B could be detected from the product. Thus, it wasconfirmed that the substitution of amino acid at position 136 of orf11created a deoxystreptamine-dependent kanamycin producing strain, S.Kanamyceticus-DOS.

Example 3 Streptamine-Added Cultivation UtilizingDeoxystreptamine-Dependent Kanamycin Producing Strain

The deoxystreptamine-dependent kanamycin producing strain obtained inExample 2 was cultivated in the liquid producing medium at 26° C. for 7days. On the second and third days, streptamine adjusted so as to have afinal concentration of 2,000 μg/ml was added.

Radio Light #800 was added to 2,000 L of the culture, and the mixturewas filtered. The filtrate was adsorbed on 50 ml-volume AmberliteFPC3500 (NH₄ ⁺ type, Rohm and Haas Japan K.K.). The resin was washedwith water, and elution was then carried out with 0.5 N aqueous ammonia.The eluate was adjusted to pH 6 and was adsorbed on 50 ml-volume Dowex50W (NH₄ ⁺ type, Muromachi Technos CO., LTD.), and elution was carriedout with 0.04 N to 0.2 N aqueous ammonia to obtain 57.6 mg of2-hydroxykanamycin A, 32.8 mg of 2-hydroxykanamycin B, and 513.2 mg of2-hydroxykanamycin C. The structures of these hydrokykanamycins weredetermined by an HR-FAB/MS (JEOL JMS-700, JEOL Ltd.) and NMR (JEOLJNM-LA400, JEOL Ltd.) spectral analysis.

Example 4 Myo-Inositol-Added Cultivation UtilizingDeoxystreptamine-Dependent Kanamycin Producing Strain

The deoxystreptamine-dependent kanamycin producing strain obtained inExample 2 was coated onto the agar medium to which myo-inositol had beenadded to a concentration of 500 μg/ml, followed by cultivation at 28° C.for 7 days. Thereafter, the agar was frozen and thawed to extract theproduct which was then subjected to an LC/MS analysis. As a result, apeak attributable to 2-hydroxykanamycin C (retention time 8.3 min, m/z501) was detected.

Example 5 Streptamine or Myo-Inositol-Added Cultivation in KanamycinProducing Bacteria

In kanamycin producing bacteria (Streptomycin kanamyceticus), agarcultivation with the addition of 500 μg/ml of streptamine ormyo-inositol was carried out in the same manner as in Example 4. Theproduct was analyzed by LC/MS. As a result, in both the addition ofstreptamine and the addition of myo-inositol, it was detected that, inaddition to kanamycin A (retention time 8.2 min, m/z 485) and kanamycinB (retention time 10.4 min, m/z 484), 2-hydroxykanamycin B (retentiontime 10.7 min, m/z 500) and 2-hydroxykanamycin C (retention time 8.3min, m/z 501) were produced.

Example 6 Confirmation of Physicochemical Properties ofHydroxykanamycins B and C

The physicochemical properties of hydroxykanamycins B and C acquired inExamples 3 to 5 were examined and were found to be as follows.

Physicochemical Properties of 2-Hydroxykanamycin B

(1) Color and properties: Colorless powder

(2) Molecular formula: C₁₈H₃₇N₅O₁₁

(3) Mass spectrum (HR-FAB/MS): measured value 500.2563 (M+H)⁺,calculated value 500.2568

(4) Specific rotation: [α]D²⁵=+127.1° (c=1, H₂O)

(5) Ultraviolet absorption spectrum λmax nm: terminal adsorption (H₂O)

(6) Infrared absorption spectrum vmax cm⁻¹ (KBr): 3351, 2910, 1585,1477, 1368, 1032

(7) ¹H-NMR spectrum (400 MHz, D₂O) δ (ppm): 2.87 (1H, dd, H-1), 3.14(1H, dd, H-2), 2.84 (1H, dd, H-3), 3.40 (1H, dd, H-4), 3.77 (1H, dd,H-5), 3.32 (1H, dd, H-6), 5.37 (1H, d, H-1′), 2.79 (1H, dd, H-2′), 3.58(1H, dd, H-3′), 3.32 (1H, dd, H-4′), 3.81 (1H, m, H-5′), 2.84 (1H, dd,H-6′a), 3.06 (1H, m, H-6′b), 5.05 (1H, d, H-1″), 3.52 (1H, dd, H-2″),3.02 (1H, dd, H-3″), 3.35 (1H, dd, H-4″), 3.93 (1H, dt, H-5″), 3.78 (2H,br d, H-6″)

[TSP=0 ppm]

(8)¹³C-NMR spectrum (100 MHz, D₂O) δ (ppm): 57.0 (d, C-1), 73.5 (d,C-2), 55.7 (d, C-3), 82.9 (d, C-4), 74.7 (d, C-5), 84.7 (d, C-6), 100.7(d, C-1′), 55.9 (d, C-2′), 74.1 (d, C-3′), 72.0 (d, C-4′), 73.3 (d,C-5′), 42.1 (t, C-6′), 100.7 (d, C-1″), 72.4 (d, C-2″), 54.9 (d, C-3″),69.8 (d, C-4″), 72.7 (d, C-5″), 60.9 (t, C-6″)

[Dioxane=67.4 ppm]

(9) Solubility: soluble in water, and insoluble in ethyl acetate andchloroform

Physicochemical Properties of 2-Hydroxykanamycin C

(1) Color and properties: Colorless powder

(2) Molecular formula: C₁₈H₃₆N₄O₁₂

(3) Mass spectrum (HR-FAB/MS): measured value 501.2398 (M+H)⁺,calculated value 501.2408

(4) Specific rotation: [α]D²⁵=+114.3° (c=1, H₂O)

(5) Ultraviolet absorption spectrum λmax nm: terminal absorption (H₂O)

(6) Infrared absorption spectrum vmax cm⁻¹ (KBr): 3358, 2920, 1591,1457, 1369, 1032

(7) ¹H-NMR spectrum (400 MHz, D₂O) δ (ppm): 2.88 (1H, dd, H-1), 3.14(1H, dd, H-2), 2.83 (1H, dd, H-3), 3.39 (1H, dd, H-4), 3.76 (1H, dd,H-5), 3.33 (1H, dd, H-6), 5.32 (1H, d, H-1′), 2.81 (1H, dd, H-2′), 3.60(1H, dd, H-3′), 3.41 (1H, dd, H-4′), 3.86 (1H, m, H-5′), 3.76 (1H, dd,H-6′a), 3.88 (1H, m, H-6′b), 5.06 (1H, d, H-1″), 3.54 (1H, dd, H-2″),3.04 (1H, dd, H-3″), 3.37 (1H, dd, H-4″), 3.94 (1H, dt, H-5″), 3.79 (2H,br d, H-6″)

[TSP=0 ppm]

(8)¹³C-NMR spectrum (100 MHz, D₂O) δ (ppm): 57.1 (d, C-1), 73.5 (d,C-2), 56.0 (d, C-3), 83.6 (d, C-4), 74.8 (d, C-5), 84.8 (d, C-6), 101.1(d, C-1′), 56.1 (d, C-2′), 74.3 (d, C-3′), 70.6 (d, C-4′), 73.7 (d,C-5′), 61.4 (t, C-6′), 100.9 (d, C-1″), 72.4 (d, C-2″), 55.1 (d, C-3″),69.7 (d, C-4″), 72.8 (d, C-5″), 60.9 (t, C-6″)

[Dioxane=67.4 ppm]

(9) Solubility: soluble in water, and insoluble in ethyl acetate andchloroform

Test Example 1 Antimicrobial Activity of 2-Hydroxykanamycin B and2-Hydroxykanamycin C

The antimicrobial activity of 2-hydroxykanamycin B and2-hydroxykanamycin C was measured as the minimum inhibitoryconcentration (MIC) by an agar dilution method. Test bacteria which hadbeen cultivated overnight in a growth medium were adjusted to 10⁶cells/ml, and one platinum loop thereof was inoculated in aMueller-Hinton agar medium containing 2-hydroxykanamycin B or2-hydroxykanamycin C (manufactured by Difco Laboratories Inc.), followedby cultivation at 37° C. for 18 to 20 hr.

The results are shown in Table 1.

TABLE 1 Antimicrobial activity of 2-hydroxykanamycins B and C MIC(μg/mL) 2-Hydroxy- 2-Hydroxy- Strain kanamycin B kanamycin CStaphylococcus aureus 2 128 ATCC29213 Staphylococcus aureus 2 128 209PJC-1 Staphylococcus epidermidis 1 32 ATCC14990 Bacillus subtilis 0.5 16ATCC6633 Enterococcus faecalis 64 >128 ATCC29212 Enterococcus faecium64 >128 ATCC19434 Salmonella typhimurium 1 32 ATCC13311 Acinetobactercalcoaceticus 1 32 ATCC23055 Escherichia coli 4 128 ATCC25922Pseudomonas aeruginosa 128 >128 PA01

1-23. (canceled)
 24. A kanamycin producing strain of the genusStreptomyces capable of producing a compound represented by formula (I),

wherein 2-deoxy-scyllo-inosose synthase has been inactivated.
 25. Thekanamycin producing strain according to claim 24 which is2-deoxystreptamine-dependent.
 26. The kanamycin producing strainaccording to claim 24, wherein a gene that codes for a polypeptideselected from the following polypeptides (a) to (d) has been integrated:(a) a polypeptide consisting of an amino acid sequence represented bySEQ ID NO:1 having a mutation in which aspartic acid at position 136 hasbeen changed to asparagine, (b) a polypeptide consisting of the aminoacid sequence defined in (a) in which one or more amino acids have beensubstituted, deleted, added, or inserted, the polypeptide having anactivity functionally equivalent to the polypeptide defined in (a), and(c) a polypeptide consisting of an amino acid sequence having 80% ormore homology with the amino acid sequence defined in (a), thepolypeptide having an activity functionally equivalent to thepolypeptide defined in (a).
 27. The strain according to claim 26,wherein the polypeptide defined in (b) or (c) holds the mutation definedin (a).
 28. The strain according to claim 26, wherein the one or moreamino acids in (b) is 1 to 40 amino acids.
 29. The strain according toclaim 26, wherein the homology in (c) is not less than 90%.
 30. Thestrain according to claim 24, wherein the strain is S.Kanamyceticus-DOS.
 31. The strain according to claim 24, capable ofproducing the compound represented by formula (I) in combination with acomponent selected from streptamine and myo-inositol.
 32. A compositioncomprising the kanamycin producing strain according to claim 24 and acomponent selected from streptamine and myo-inositol.
 33. A compositioncomprising the kanamycin producing strain according to claim 25 and acomponent selected from streptamine and myo-inositol.
 34. A compositioncomprising the kanamycin producing strain according to claim 26 and acomponent selected from streptamine and myo-inositol.
 35. A compositioncomprising the kanamycin producing strain according to claim 27 and acomponent selected from streptamine and myo-inositol.
 36. A compositioncomprising the kanamycin producing strain according to claim 28 and acomponent selected from streptamine and myo-inositol.
 37. A compositioncomprising the kanamycin producing strain according to claim 29 and acomponent selected from streptamine and myo-inositol.
 38. A compositioncomprising the kanamycin producing strain according to claim 30 and acomponent selected from streptamine and myo-inositol.
 39. A compositioncomprising the kanamycin producing strain according to claim 31 and acomponent selected from streptamine and myo-inositol.
 40. A method fortreating or preventing an infectious disease, comprising administeringan effective amount of a compound represented by formula (I) or itspharmacologically acceptable salt or their solvates to an animalincluding human:

wherein R represents amino or hydroxyl.
 41. The process according toclaim 40, wherein the infectious disease is derived from Staphylococcusaureus, Escherichia coli, or Pseudomonas aeruginosa.